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1.
Cell ; 150(5): 1055-67, 2012 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-22939628

RESUMO

The type 2 ryanodine receptor/calcium release channel (RyR2), required for excitation-contraction coupling in the heart, is abundant in the brain. Chronic stress induces catecholamine biosynthesis and release, stimulating ß-adrenergic receptors and activating cAMP signaling pathways in neurons. In a murine chronic restraint stress model, neuronal RyR2 were phosphorylated by protein kinase A (PKA), oxidized, and nitrosylated, resulting in depletion of the stabilizing subunit calstabin2 (FKBP12.6) from the channel complex and intracellular calcium leak. Stress-induced cognitive dysfunction, including deficits in learning and memory, and reduced long-term potentiation (LTP) at the hippocampal CA3-CA1 connection were rescued by oral administration of S107, a compound developed in our laboratory that stabilizes RyR2-calstabin2 interaction, or by genetic ablation of the RyR2 PKA phosphorylation site at serine 2808. Thus, neuronal RyR2 remodeling contributes to stress-induced cognitive dysfunction. Leaky RyR2 could be a therapeutic target for treatment of stress-induced cognitive dysfunction.


Assuntos
Transtornos Cognitivos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Cálcio/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos de Estresse Traumático/metabolismo
2.
Int J Mol Sci ; 25(6)2024 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-38542361

RESUMO

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes raise the possibility of generating pluripotent stem cells from a wide range of human diseases. In the cardiology field, hiPSCs have been used to address the mechanistic bases of primary arrhythmias and in investigations of drug safety. These studies have been focused primarily on atrial and ventricular pathologies. Consequently, many hiPSC-based cardiac differentiation protocols have been developed to differentiate between atrial- or ventricular-like cardiomyocytes. Few protocols have successfully proposed ways to obtain hiPSC-derived cardiac pacemaker cells, despite the very limited availability of human tissues from the sinoatrial node. Providing an in vitro source of pacemaker-like cells would be of paramount importance in terms of furthering our understanding of the mechanisms underlying sinoatrial node pathophysiology and testing innovative clinical strategies against sinoatrial node dysfunction (i.e., biological pacemakers and genetic- and pharmacological- based therapy). Here, we summarize and detail the currently available protocols used to obtain patient-derived pacemaker-like cells.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Miócitos Cardíacos , Diferenciação Celular/fisiologia , Nó Sinoatrial
3.
Bioinformatics ; 37(22): 4209-4215, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34048539

RESUMO

MOTIVATION: Cardiomyocytes derived from stem cells are closely followed, notably since the discovery in 2007 of human induced pluripotent stem cells (hiPSC). Cardiomyocytes (hiPSC-CM) derived from hiPSC are indeed more and more used to study specific cardiac diseases as well as for developing novel applications such as drug safety experiments. Robust dedicated tools to characterize hiPSC-CM are now required. The hiPSC-CM morphology constitutes an important parameter since these cells do not demonstrate the expected rod shape, characteristic of native human cardiomyocytes. Similarly, the presence, the density and the organization of contractile structures would be a valuable parameter to study. Precise measurements of such characteristics would be useful in many situations: for describing pathological conditions, for pharmacological screens or even for studies focused on the hiPSC-CM maturation process. RESULTS: For this purpose, we developed a MATLAB based image analysis toolbox, which gives accurate values for cellular morphology parameters as well as for the contractile cell organization. AVAILABILITY AND IMPLEMENTATION: To demonstrate the power of this automated image analysis, we used a commercial maturation medium intended to promote the maturation status of hiPSC-CM, and compare the parameters with the ones obtained with standard culture medium, and with freshly dissociated mouse cardiomyocytes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Animais , Camundongos , Células Cultivadas
4.
Pflugers Arch ; 473(7): 1099-1115, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34169350

RESUMO

Duchenne muscular dystrophy is a genetic disorder where an X-linked mutation in the DMD gene initiates pathogenic development caused by the absence of dystrophin protein. This impacts primarily the evolution of a functional muscle tissue resulting in muscle weakness and later severe disability in young male patients leading to an early death. Patients in the final stage develop dilated cardiomyopathy leading ultimately to cardiac or respiratory failure as the cause of death. This review discusses recent advances in modeling the DMD pathology in vitro. It describes in detail the molecular abnormalities found on the cellular and organoid levels. The in vitro pathology is compared to that found in patients. Likewise, the drawbacks and limitations of current models are discussed.


Assuntos
Coração/fisiologia , Distrofia Muscular de Duchenne/patologia , Animais , Cardiomiopatia Dilatada/patologia , Humanos
5.
Int J Mol Sci ; 22(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440843

RESUMO

Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.


Assuntos
Diferenciação Celular , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Oxigênio/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Biomarcadores , Cálcio/metabolismo , Técnicas de Cultura de Células , Expressão Gênica , Humanos , Mitocôndrias Cardíacas/metabolismo , Retículo Sarcoplasmático/metabolismo , Esferoides Celulares
6.
Int J Mol Sci ; 22(23)2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34884796

RESUMO

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting following repeated muscle damage and inadequate regeneration. Impaired myogenesis and differentiation play a major role in DMD as well as intracellular calcium (Ca2+) mishandling. Ca2+ release from the sarcoplasmic reticulum is mostly mediated by the type 1 ryanodine receptor (RYR1) that is required for skeletal muscle differentiation in animals. The study objective was to determine whether altered RYR1-mediated Ca2+ release contributes to myogenic differentiation impairment in DMD patients. The comparison of primary cultured myoblasts from six boys with DMD and five healthy controls highlighted delayed myoblast differentiation in DMD. Silencing RYR1 expression using specific si-RNA in a healthy control induced a similar delayed differentiation. In DMD myotubes, resting intracellular Ca2+ concentration was increased, but RYR1-mediated Ca2+ release was not changed compared with control myotubes. Incubation with the RYR-calstabin interaction stabilizer S107 decreased resting Ca2+ concentration in DMD myotubes to control values and improved calstabin1 binding to the RYR1 complex. S107 also improved myogenic differentiation in DMD. Furthermore, intracellular Ca2+ concentration was correlated with endomysial fibrosis, which is the only myopathologic parameter associated with poor motor outcome in patients with DMD. This suggested a potential relationship between RYR1 dysfunction and motor impairment. Our study highlights RYR1-mediated Ca2+ leakage in human DMD myotubes and its key role in myogenic differentiation impairment. RYR1 stabilization may be an interesting adjunctive therapeutic strategy in DMD.


Assuntos
Desenvolvimento Muscular/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Distrofia Muscular de Duchenne/patologia , Mioblastos/citologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Criança , Pré-Escolar , Distrofina/metabolismo , Humanos , Masculino , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular de Duchenne/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo
7.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068508

RESUMO

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.


Assuntos
Cardiomiopatia Dilatada/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Envelhecimento/genética , Envelhecimento/patologia , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Dano ao DNA/genética , Modelos Animais de Doenças , Distrofina/deficiência , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Células-Tronco/metabolismo , Células-Tronco/patologia
8.
Nanomedicine ; 30: 102295, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32889047

RESUMO

Duchenne muscular dystrophy (DMD) is a debilitating disorder related to dystrophin encoding gene mutations, often associated with dilated cardiomyopathy. However, it is still unclear how dystrophin deficiency affects cardiac sarcomere remodeling and contractile dysfunction. We employed second harmonic generation (SHG) microscopy, a nonlinear optical imaging technique that allows studying contractile apparatus organization without histologic fixation and immunostaining. Images were acquired on alive DMD (mdx) and wild type cardiomyocytes at different ages and at various external calcium concentrations. An automated image processing was developed to identify individual myofibrils and extract data about their organization. We observed a structural aging-dependent remodeling in mdx cardiomyocytes affecting sarcomere sinuosity, orientation and length that could not be anticipated from standard optical imaging. These results revealed for the first time the interest of SHG to evaluate the intracellular and sarcomeric remodeling of DMD cardiac tissue in an age-dependent manner that could participate in progressive contractile dysfunction.


Assuntos
Distrofina/genética , Miócitos Cardíacos/metabolismo , Microscopia de Geração do Segundo Harmônico/métodos , Animais , Senescência Celular , Camundongos , Camundongos Endogâmicos C57BL , Distrofia Muscular de Duchenne/genética , Miócitos Cardíacos/patologia , Sarcômeros/patologia
9.
J Mol Cell Cardiol ; 131: 155-163, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31051181

RESUMO

The mechanical and cellular relationships between systole and diastole during left ventricular (LV) dysfunction remain to be established. LV contraction-relaxation coupling was examined during LV hypertrophy induced by chronic hypertension. Chronically instrumented pigs received angiotensin II infusion for4weeks to induce chronic hypertension (133 ±â€¯7 mmHg vs 98 ±â€¯5 mmHg for mean arterial pressure at Day 28 vs 0, respectively) and LV hypertrophy. LV function was investigated with the instrumentation and echocardiography for LV twist-untwist assessment before and after dobutamine infusion. The cellular mechanisms were investigated by exploring the intracellular Ca2+ handling. At Day 28, pigs exhibited LV hypertrophy with LV diastolic dysfunction (impaired LV isovolumic relaxation, increased LV end-diastolic pressure, decreased and delayed LV untwisting rate) and LV systolic dysfunction (impaired LV isovolumic contraction and twist) although LV ejection fraction was preserved. Isolated cardiomyocytes exhibited altered shortening and lengthening. Interestingly, contraction-relaxation coupling remained preserved both in vivo and in vitro during LV hypertrophy. LV systolic and diastolic dysfunctions were associated to post-translational remodeling and dysfunction of the type 2 cardiac ryanodine receptor/Ca2+ release channel (RyR2), i.e., PKA hyperphosphorylation of RyR2, depletion of calstabin 2 (FKBP12.6), RyR2 leak and hypersensitivity of RyR2 to cytosolic Ca2+ during both contraction and relaxation phases. In conclusion, LV contraction-relaxation coupling remained preserved during chronic hypertension despite LV systolic and diastolic dysfunctions. This implies that LV diastolic dysfunction is accompanied by LV systolic dysfunction. At the cellular level, this is linked to sarcoplasmic reticulum Ca2+ leak through PKA-mediated RyR2 hyperphosphorylation and depletion of its stabilizing partner.


Assuntos
Diástole/fisiologia , Hipertensão/fisiopatologia , Sístole/fisiologia , Animais , Western Blotting , Ecocardiografia , Frequência Cardíaca/fisiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Imunoprecipitação , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Suínos , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular Esquerda/fisiologia
10.
Acta Neuropathol ; 134(5): 749-767, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28631094

RESUMO

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.


Assuntos
Doença de Alzheimer/metabolismo , Cálcio/metabolismo , Transtornos Cognitivos/metabolismo , Processamento de Proteína Pós-Traducional , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Doença de Alzheimer/patologia , Animais , Sinalização do Cálcio , Transtornos Cognitivos/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/fisiologia , Fosforilação , Reconhecimento Psicológico/fisiologia , Retículo Sarcoplasmático/metabolismo
12.
Mol Microbiol ; 98(3): 490-501, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26192332

RESUMO

Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane ß barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner ß-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the ß barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Heart Vessels ; 29(6): 834-46, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24258387

RESUMO

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and ß, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca(2+) imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca(2+) from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the ß-adrenergic pathway.


Assuntos
Corpos Embrioides/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos , Retículo Sarcoplasmático/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular , Forma Celular , Tamanho Celular , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Reprodutibilidade dos Testes , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Troponina T/metabolismo
14.
Proc Natl Acad Sci U S A ; 108(32): 13258-63, 2011 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-21788490

RESUMO

Myocardial ischemic disease is the major cause of death worldwide. After myocardial infarction, reperfusion of infracted heart has been an important objective of strategies to improve outcomes. However, cardiac ischemia/reperfusion (I/R) is characterized by inflammation, arrhythmias, cardiomyocyte damage, and, at the cellular level, disturbance in Ca(2+) and redox homeostasis. In this study, we sought to determine how acute inflammatory response contributes to reperfusion injury and Ca(2+) homeostasis disturbance after acute ischemia. Using a rat model of I/R, we show that circulating levels of TNF-α and cardiac caspase-8 activity were increased within 6 h of reperfusion, leading to myocardial nitric oxide and mitochondrial ROS production. At 1 and 15 d after reperfusion, caspase-8 activation resulted in S-nitrosylation of the RyR2 and depletion of calstabin2 from the RyR2 complex, resulting in diastolic sarcoplasmic reticulum (SR) Ca(2+) leak. Pharmacological inhibition of caspase-8 before reperfusion with Q-LETD-OPh or prevention of calstabin2 depletion from the RyR2 complex with the Ca(2+) channel stabilizer S107 ("rycal") inhibited the SR Ca(2+) leak, reduced ventricular arrhythmias, infarct size, and left ventricular remodeling after 15 d of reperfusion. TNF-α-induced caspase-8 activation leads to leaky RyR2 channels that contribute to myocardial remodeling after I/R. Thus, early prevention of SR Ca(2+) leak trough normalization of RyR2 function is cardioprotective.


Assuntos
Caspase 8/metabolismo , Ventrículos do Coração/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Ativação Enzimática , Fluorescência , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Fenantridinas/metabolismo , Ratos , Ratos Endogâmicos WKY , Fator de Necrose Tumoral alfa/sangue , Remodelação Ventricular
15.
J Cachexia Sarcopenia Muscle ; 15(2): 536-551, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38221511

RESUMO

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle weakness due to the absence of functional dystrophin. DMD patients also develop dilated cardiomyopathy (DCM). We have previously shown that DMD (mdx) mice and a canine DMD model (GRMD) exhibit abnormal intracellular calcium (Ca2+) cycling related to early-stage pathological remodelling of the ryanodine receptor intracellular calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) contributing to age-dependent DCM. METHODS: Here, we used hiPSC-CMs from DMD patients selected by Speckle-tracking echocardiography and canine DMD cardiac biopsies to assess key early-stage Duchenne DCM features. RESULTS: Dystrophin deficiency was associated with RyR2 remodelling and SR Ca2+ leak (RyR2 Po of 0.03 ± 0.01 for HC vs. 0.16 ± 0.01 for DMD, P < 0.01), which led to early-stage defects including senescence. We observed higher levels of senescence markers including p15 (2.03 ± 0.75 for HC vs. 13.67 ± 5.49 for DMD, P < 0.05) and p16 (1.86 ± 0.83 for HC vs. 10.71 ± 3.00 for DMD, P < 0.01) in DMD hiPSC-CMs and in the canine DMD model. The fibrosis was increased in DMD hiPSC-CMs. We observed cardiac hypocontractility in DMD hiPSC-CMs. Stabilizing RyR2 pharmacologically by S107 prevented most of these pathological features, including the rescue of the contraction amplitude (1.65 ± 0.06 µm for DMD vs. 2.26 ± 0.08 µm for DMD + S107, P < 0.01). These data were confirmed by proteomic analyses, in particular ECM remodelling and fibrosis. CONCLUSIONS: We identified key cellular damages that are established earlier than cardiac clinical pathology in DMD patients, with major perturbation of the cardiac ECC. Our results demonstrated that cardiac fibrosis and premature senescence are induced by RyR2 mediated SR Ca2+ leak in DMD cardiomyocytes. We revealed that RyR2 is an early biomarker of DMD-associated cardiac damages in DMD patients. The progressive and later DCM onset could be linked with the RyR2-mediated increased fibrosis and premature senescence, eventually causing cell death and further cardiac fibrosis in a vicious cycle leading to further hypocontractility as a major feature of DCM. The present study provides a novel understanding of the pathophysiological mechanisms of the DMD-induced DCM. By targeting RyR2 channels, it provides a potential pharmacological treatment.


Assuntos
Cardiomiopatias , Cardiomiopatia Dilatada , Humanos , Camundongos , Animais , Cães , Cardiomiopatia Dilatada/etiologia , Distrofina/genética , Distrofina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Camundongos Endogâmicos mdx , Cálcio/metabolismo , Proteômica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fibrose
16.
Circ Res ; 109(3): 281-90, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21659649

RESUMO

RATIONALE: Mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT-associated RyR2 mutations cause fatal ventricular arrhythmias in young individuals during ß-adrenergic stimulation. OBJECTIVE: This study sought to determine the effects of a novel RyR2-G230C mutation and whether this mutation and RyR2-P2328S alter the sensitivity of the channel to luminal calcium (Ca(2+)). METHODS AND RESULTS: Functional characterizations of recombinant human RyR2-G230C channels were performed under conditions mimicking stress. Human RyR2 mutant channels were generated by site-directed mutagenesis and heterologously expressed in HEK293 cells together with calstabin2. RyR2 channels were measured to examine the regulation of the channels by cytosolic versus luminal sarcoplasmic reticulum Ca(2+). A 50-year-old white man with repeated syncopal episodes after exercise had a cardiac arrest and harbored the mutation RyR2-G230C. cAMP-dependent protein kinase-phosphorylated RyR2-G230C channels exhibited a significantly higher open probability at diastolic Ca(2+) concentrations, associated with a depletion of calstabin2. The luminal Ca(2+) sensitivities of RyR2-G230C and RyR2-P2328S channels were WT-like. CONCLUSIONS: The RyR2-G230C mutant exhibits similar biophysical defects compared with previously characterized CPVT mutations: decreased binding of the stabilizing subunit calstabin2 and a leftward shift in the Ca(2+) dependence for activation under conditions that simulate exercise, consistent with a "leaky" channel. Both RyR2-G230C and RyR2-P2328S channels exhibit normal luminal Ca(2+) activation. Thus, diastolic sarcoplasmic reticulum Ca(2+) leak caused by reduced calstabin2 binding and a leftward shift in the Ca(2+) dependence for activation by diastolic levels of cytosolic Ca(2+) is a common mechanism underlying CPVT.


Assuntos
Cálcio/fisiologia , Morte Súbita Cardíaca , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Taquicardia Ventricular/fisiopatologia , Catecolaminas/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Citosol/fisiologia , Diástole/fisiologia , Eletrocardiografia , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Masculino , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Fenótipo , Mutação Puntual , Proteínas Recombinantes/genética , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/genética , Proteínas de Ligação a Tacrolimo/fisiologia
17.
Stem Cell Res ; 72: 103192, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37660555

RESUMO

Type 2 Long QT Syndrome (LQT2) is a rare genetic heart rhythm disorder causing life-threatening arrhythmias. We derived induced pluripotent stem cell (iPSC) lines from two patients with LQT2, aged 18 and 6, both carrying a heterozygous missense mutation on the 3rd and 11th exons of KCNH2. The iPSC lines exhibited normal genomes, expressed pluripotent markers, and differentiated into trilineage embryonic layers. These patient-specific iPSC lines provide a valuable model to study the molecular and functional impact of the hERG channel gene mutation in LQT2 and to develop personalized therapeutic approaches for this syndrome.


Assuntos
Células-Tronco Pluripotentes Induzidas , Síndrome do QT Longo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Canal de Potássio ERG1/genética , Síndrome do QT Longo/metabolismo , Arritmias Cardíacas/metabolismo , Mutação
18.
Stem Cell Res Ther ; 14(1): 266, 2023 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-37740238

RESUMO

BACKGROUND: Polymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca2+ handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro. METHODS: Previously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs. RESULTS: The voltage-dependent Ca2+ channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca2+ in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control. CONCLUSIONS: By testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca2+ release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest.


Assuntos
Cálcio , Taquicardia Ventricular , Humanos , Miócitos Cardíacos , Flecainida/farmacologia , Propranolol/farmacologia , Propranolol/uso terapêutico , Antiarrítmicos , Medicina de Precisão , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/tratamento farmacológico , Taquicardia Ventricular/genética , Verapamil/farmacologia , Verapamil/uso terapêutico
19.
Front Cell Dev Biol ; 10: 1030950, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36274847

RESUMO

Cardiomyopathy is a myocardial disorder, in which the heart muscle is structurally and functionally abnormal, often leading to heart failure. Dilated cardiomyopathy is characterized by a compromised left ventricular function and contributes significantly to the heart failure epidemic, which represents a staggering clinical and public health problem worldwide. Gene mutations have been identified in 35% of patients with dilated cardiomyopathy. Pathogenic variants in LMNA, encoding nuclear A-type lamins, are one of the major causative causes of dilated cardiomyopathy (i.e. CardioLaminopathy). A-type lamins are type V intermediate filament proteins, which are the main components of the nuclear lamina. The nuclear lamina is connected to the cytoskeleton on one side, and to the chromatin on the other side. Among the models proposed to explain how CardioLaminopathy arises, the "chromatin model" posits an effect of mutated A-type lamins on the 3D genome organization and thus on the transcription activity of tissue-specific genes. Chromatin contacts with the nuclear lamina via specific genomic regions called lamina-associated domains lamina-associated domains. These LADs play a role in the chromatin organization and gene expression regulation. This review focuses on the identification of LADs and chromatin remodeling in cardiac muscle cells expressing mutated A-type lamins and discusses the methods and relevance of these findings in disease.

20.
Stem Cell Res ; 60: 102727, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35245853

RESUMO

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is a genetic disorder characterized by ventricular tachycardia, that can cause the heart to stop beating leading to death. The prevalence is 1/10.000 and in approximately 60% of cases, the syndrome can be due to a mutation of the cardiac ryanodine receptor gene (RyR2). We derived an induced pluripotent stem cell (iPSC) line from an 11-year-old patient blood-cells, carrying a heterozygous missense mutation on the 8th exon of the RyR2 N-terminal part. This reprogramed CPVT line displayed normal karyotype, expressed pluripotent markers and had a capacity to differentiate in trilineage embryonic layers.


Assuntos
Células-Tronco Pluripotentes Induzidas , Taquicardia Ventricular , Criança , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/genética
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